Chikungunya virus (CHIKV) is a positive-strand RNA virus of the genus Alphavirus within the family of Togaviridae, first isolated in Tanzania in 1952.
Infection by this virus causes human disease that is characterized by symptoms similar to dengue fever, with an acute febrile phase during two to five days, followed by a prolonged arthralgic disease that affects the joints of the extremities. CHIKV is endemic in Africa, India and South-East Asia and is transmitted by Aedes mosquitoes through an urban or sylvatic transmission cycle. In 2006, an outbreak of CHIKV fever occurred in numerous islands of the Indian Ocean (the Comoros, Mauritius, Seychelles, Madagascar, Reunion island . . . ), before jumping to India where an estimated 1.4 million cases have been reported. More recently, imported infections have been described in Europe, and around 200 endemic cases have been reported in Italy (Jose, J. et al., A structural and functional perspective of alphavirus replication and assembly. Future Microbiol, 2009. 4(7): p. 837-56). Clinically, this CHIKV epidemic was accompanied by more severe symptoms than previous outbreaks, with reports of severe polyarthralgia and myalgia, complications and deaths.
The CHIKV genome is an 11.8 kb, single-stranded RNA molecule of positive polarity. This virus is closely related to Semliki Forest virus (SFV), Sindbis virus (SINV), and other Old-World alphaviruses, and more distantly related to New-World alphaviruses like Venezuelan equine encephalitis virus (Griffin, D. E., Alphaviruses, in Fields Virology, 5th ed., D. M. Knipe, Editor 2007, Wolters Kluwer, Lippincott Williams & Wilkins. p. 1023-1067). The genomic RNA is capped, and directly translated into a full-length non-structural polyprotein (nsP) called P1234, which is encoded by the 5′ two-thirds of the genome (Jose, J., J. E. Snyder, and R. J. Kuhn, A structural and functional perspective of alphavirus replication and assembly. Future Microbiol, 2009. 4(7): p. 837-56; Kuhn, R. J., Togaviridae: the viruses and their replication, in Fields Virology, 5th ed., D. M. Knipe, Editor 2007, Wolters Kluwer, Lippincott Williams & Wilkins. p. 1001-1022). This precursor cleaves itself to produce P123 and nsP4 that carries the RNA-dependent RNA polymerase activity. These proteins, together with cellular co-factors, assemble into a replication complex that produces antisense genomic RNA molecules. Subsequent cleavage of P123 into nsP1 and P23 gives rise to a polymerase complex making both sense and antisense genomic RNA. Further processing of P23 into nsP2 and nsP3 gives rise to a polymerase complex making only positive-sense genomic RNA molecules. In addition to replicating the viral genome, this viral protein complex transcribes a 26S subgenomic RNA from the 3′ extremity of the viral genome. This messenger RNA is translated into a polyprotein precursor, which is cleaved by a combination of viral and cellular enzymes to produce a capsid protein (C), two major envelope proteins (E1 and E2), and two smaller accessory peptides, E3 and 6k. Once assembled, CHIKV virions are spherical particles of 65-70 nm in diameter, essentially composed of genomic RNA molecules associated with capsid proteins, and enveloped in a host-derived lipid membrane decorated by E1-E2 heterodimers organized in an icosahedral lattice (Voss, J. E., et al., Glycoprotein organization of Chikungunya virus particles revealed by X-ray crystallography. Nature, 2010. 468(7324): p. 709-12).
The disease severity, as much as the evolution and spread of the virus into new geographic areas are a serious health public matter that needs to be fixed. In order to solve this problem, vaccines with live attenuated virus, with chimeric alphavirus, with recombinant DNA or with virus-like particles have been developed.
A formalin inactivated vaccine was shown to be immunogenic in non-human primates and humans, but the high amount of antigen required for mass immunization that needs to be prepared under BSL-3 conditions is a limitation for the development of this strategy (Tiwari, M., et al., Assessment of immunogenic potential of Vero adapted formalin inactivated vaccine derived from novel ECSA genotype of Chikungunya virus. (Vaccine, 2009. 27(18): p. 2513-22). The live attenuated TSI-GSD-218 CHIKV vaccine developed by the US-Army was immunogenic but caused side effects in Phase II clinical trials associated with reversion to virulence raising safety issues. Therefore, although the results obtained with vaccines based on live attenuated virus show that an efficient immunization can be achieved by this way, those vaccines are still questionable as there could be a risk of possibly side effects (Edelman R et al., Am J Trop Med Hyg. 2000 June; 62(6):681-685). Chimeric alphavirus vaccine strategies encoding the E1, E2 and capsid proteins from CHIKV are immunogenic in mice (Wang, E., et al., Chimeric alphavirus vaccine candidates for Chikungunya. Vaccine, 2008. 26(39): p. 5030-9), but the ability of alphavirus to easily recombine raises safety issues against the development of such strategies (Weaver, S. C., et al., Recombinational history and molecular evolution of western equine encephalomyelitis complex alphaviruses. J Virol, 1997. 71(1): p. 613-23).
Another strategy, which has been explored is to design recombinant DNA construct for use as a vaccine. DNA based CHIKV vaccines encoding the E1, E2 and capsid protein have been shown to be immunogenic in mice and non-human primates (Muthumani, K., et al., Immunogenicity of novel consensus-based DNA vaccines against Chikungunya virus. Vaccine, 2008. 26(40): p. 5128-34; Mallilankaraman, K., et al., A DNA vaccine against chikungunya virus is protective in mice and induces neutralizing antibodies in mice and nonhuman primates. PLoS Negl Trop Dis, 2011. 5(1): p. e928), but the DNA strategies do not induce the strong neutralizing immune response required for CHIKV clearance in humans. The disadvantages of DNA vaccines are that high quantities of DNA are required to induce an immune response and multiple booster vaccinations must be performed. The need for multiple boosts and high quantities of DNA injected into the nuclei of many cells raises concern regarding the fact that DNA vaccines can integrate into the host DNA and cause insertional mutagenesis. Therefore a recent study reports using DNA vaccines combined with live attenuated virus (WO2011/082388). Although this technique allows reducing drawbacks of live attenuated virus and DNA vaccines, there is still a need in providing a vaccine with reduced side effects.
In order to avoid drawbacks of live attenuated virus and DNA vaccines, other types of vaccines have been developed such as vaccines based on virus-like particles (VLPs) which are obtained by expressing structural proteins of the Chikungunya virus. These structural proteins are able to self-assemble in virus-like particles. On that basis, vaccines comprising polynucleotides encoding all Chikungunya virus structural proteins have been worked out (Akahata, W., et al., A virus-like particle vaccine for epidemic Chikungunya virus protects nonhuman primates against infection. Nat Med, 2010. 16(3): p. 334-8). However, VLPs produced in vitro are expensive to manufacture and require three administrations for a complete immunity, therefore these vaccines are not cheaply affordable. The CHIKV VLPs strategy disclosed in Akata et al. required several immunizations with an adjuvant to induce protection. For this reason, there is still a need for the design of improved vaccines that would enable the CHIKV VLPs to be generated in vivo in infected cells, in particular in infected cells of a host, and thus to provide an efficient and long-lasting immunity, especially which induces life-long immunity after only a single or two administration steps.